Abstract Graft versus host disease (GVHD) is the primary cause of morbidity and mortality after hematopoietic cell transplantation (HCT) affecting up to 70% of patients and directly accounting for a third of regimen-related death. Standard prophylaxis has not changed in over 30 years and especially promising new agents are lacking. Mesenchymal stromal cells (MSCs), with immune suppressive properties, have been touted as therapy for GVHD, but clinical trials have generated conflicting results across studies raising doubt of their utility. In our view, the fundamental barrier to the successful clinical development of MSCs, the recent study of Galleu et al. notwithstanding, is that we do not have a unified model of the mechanisms of MSC-mediated immune suppression in vivo. We have recently shown that interferon ? primed MSCs (?MSCs), which traffic to secondary lymphoid organs (SLO) after HCT, are phagocytes that engulf alloreactive T cells by efferocytosis. This process is essential for suppression of T cell proliferation and prevention of lethal GVHD in a murine model. The suppressive effect is dependent on (i) trafficking to secondary lymphoid organs (SLO), (ii) ?MSC dose and (iii) potential to engulf T cells. Thus, we posit to enhance the potency of ?MSC prophylaxis of GVHD, we must enhance the potency of ?MSC efferocytosis. The overarching objective of this proposal is to identify actionable targets in the molecular mechanism of ?MSC efferocytosis that can be leveraged to increase the potency of ?MSC engulfment, and thereby, suppression of GVHD. In aim 1, we will identify the chemokines and corresponding receptors that govern ?MSC trafficking to SLO. Then, we will enforce expression of the relevant receptors in ?MSCs to determine if that will increase the efficiency of trafficking resulting in great SLO localization, greater T cell suppression, and more effective GVHD prophylaxis. In Aim 2, we will determine the extent to which Axl/Gas6 mediates the binding and engulfment of activated T cells. Additionally, we will determine if enforced Axl expression enhances binding and engulfment and thereby the potency of ?MSC suppression of GVHD. In Aim 3, we will determine the extent to which ?MSCs engage LC3-associated phagocytosis (LAP) to engulf activated T cells. LAP is a form of noncanonical autophagy that can be stimulated by sirolimus (rapamycin). If LAP is the primary mechanism of T cell engulfment, then concomitant therapy with sirolimus could potentiate ?MSC suppression of GVHD. The data generated in this 5-year proposal has the potential to impact all patients undergoing HCT and possibly expand the eligibility for HCT by decreasing the risk of regimen-related morbidity and mortality.